Diagnosis of histoplasmosis using antigens specific for H. capsulatum

ABSTRACT

A method for identifying a protein antigen to a target fungus is disclosed. The method comprises screening expressed proteins from a cDNA gene expression library with antisera to the target fungus and cross-screening with antisera to a nontarget fungus. Antibodies to the protein antigen are also disclosed. Methods for detecting the presence or absence of the antibodies or of the protein antigen are also disclosed, as well as kits for performing such assays. In preferred embodiments, the target fungus is  H. capsulatum.

This application claims priority from U.S. Provisional ApplicationSerial No. 60/043,332, filed Apr. 15, 1997, which is incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The present invention generally relates to methods of detection of afungus and fungal infections through the use of protein antigensspecific for the fungus. This invention also relates to methods foridentifying such antigens, to antibodies raised against these antigens,to the use of such antigens and/or antibodies in assay methods for thedetermination of the presence of the fungus or diseases caused by thefungus in humans, other animals, plants, food, feed, and inorganicmaterials such as soil or air. The invention also relates to assay kitssuitable for carrying out such diagnostic methods. In a preferredembodiment, the invention particularly relates to the fungus Histoplasmacapsulatum (hereinafter H. capsulatum) and the infections caused by thisfungus, histoplasmosis.

Rapid, positive detection of fungi has long been difficult becauseantibodies which are made to fungi are generally nonspecific. That is,these antibodies often cross-react with other fungi. This problem isthought to be due to the abundance of highly immunogenic carbohydrateantigenic determinants which are present, for example, in the fungalcell wall. The cross-reactive nature of fungal antigens is exemplifiedby previously known approaches to develop diagnostic methods to identifyinfections of H. capsulatum. H. capsulatum is a pathogenic dimorphicfungus that grows as multicellular mycelia in nature and as unicellularbudding yeasts in humans and animals. Inhalation of airborne propagulesresults in a morphological transformation to the yeast form which causespulmonary infection and occasional progressive disease, particularly inimmunosuppressed patients. Histoplasmosis is highly endemic in the Ohioand Mississippi valleys in the United States, and it is also widelydistributed in Latin America, southern Europe, Asia, Australia andAfrica.

The diagnosis of histoplasmosis in humans is often suggested by resultsof a careful clinical evaluation and radiologic studies, but laboratorytests are necessary to confirm the diagnosis. Isolation of the organismfrom blood or tissue provides a definitive diagnosis. Serological testsare also important diagnostic tools for histoplasmosis. The most widelyavailable tests are the immunodiffusion assay, which detects antibodiesto heat-sensitive glycoproteins called H and M antigens, and the moresensitive complement fixation test, which is traditionally performedwith yeast and mycelial antigens. More sensitive antibody assays such asradio-immunoassay and enzyme immunoassay have been used to detect IgMand IgG antibodies to crude fungal extracts. See, generally, references39, 40, and 41.

Attempts to develop antibody serology tests for diagnosis ofhistoplasmosis have been hampered by poor specificity caused byimmunologic cross-reactivity between various fungal species. The problemof cross-reactivity to other fungi may be worsened in the diagnosis ofhistoplasmosis by the fact that H. capsulatum is taxonomically closelyrelated to two other pathogens, Coccidioides immitis, and Blastomycesdermatitidis. See Kwon-Chung, Science 177:368-369 (1972), and McGinniset al., Mycotaxon 8:157-164 (1979). These fungi, which may be presentalong with H. capsulatum, cause coccidiomycosis and blastomycosis,diseases with etiologies similar to histoplasmosis. While H. capsulatum,C. immitis, and B. dermatitidis are known as imperfect fungi due totheir rare or nonexistent sexual stage, studies have shown that H.capsulatum and B. dermatitidis are in the same telomorph genus,Ajellomyces, and those two fungi may be in the same taxonomic family asC. immitis, the Gymnoascaceae, order Onygenales, of the Ascomycetes.Another fungal pathogen, Candida sp., is also an ascomycete. Seegenerally, Kwan-Chung et al., Medical Mycology, Lea and Febiger,Philadelphia (1992), which is incorporated by reference.

In view of the cross-reactivity and poor specificity common with fungalantibodies, there is a need for improved methods for identifyingantigens which are specific to a target fungus. Such antigens are usefulin diagnosis of diseases caused by the fungus and in determining thepresence or absence of the fungus.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to develop methods suitablefor identifying protein antigens specific to a target fungus and,particularly, to H. capsulatum.

It is also an object to develop assays and assay reagents havingimproved specificity for identifying target fungal antigens andantibodies, and for the diagnosis of diseases caused by a target fungusand, particularly, by H. capsulatum.

Therefore, the present invention is directed to a method for identifyinga protein antigen of a target fungus. A cDNA gene expression library isobtained for the target fungus, and the library is expressed to form anarray of target-fungus proteins. Antisera to the target fungus and to anontarget fungus are also obtained, each of which comprises antibodiesto the target fungus and nontarget fungus, respectively. The nontargetfungus has at least one antigenic determinant (e.g. a proteindeterminant or glycoprotein determinant) in common (i.e., shared with)the target fungus. A protein antigen specific to the target fungus isthen identified by identifying a target-fungus protein which is bound bythe antibodies to the target fungus, but which is not substantiallybound by antibodies to the nontarget fungus. That is, while antibodiesto the target fungus are immunoreactive with the identified proteinantigen, antibodies to the nontarget fungus are not substantiallyimmunoreactive with the identified protein antigen.

The invention is also directed to substantially purified or isolatedantibodies or antibody fragments. In one embodiment, the antibody orantibody fragment is immunoreactive with a protein antigen identifiedaccording to the aforementioned method. In another embodiment, theantibody or antibody fragment is immunoreactive with an antigen of H.capsulatum, but which is not substantially immunoreactive with antigensof each of Coccidioides immitis, Blastomyces dermatitidis or Candida sp.In a further embodiment, antibody or antibody fragment is immunoreactivewith a protein antigen having the amino acid sequence set forth in SEQID NO: 3 or with a portion thereof that is specific to H. capsulatum.

The invention is directed, moreover, to a method for determining thepresence or absence of a target-fungus antibody in a vertebrate such asa mammal. In one embodiment, the method comprises obtaining anantibody-containing sample from the vertebrate, contacting the samplewith a target-fungus protein antigen, and determining whether anantibody in the sample immunoreacts with the target-fungus proteinantigen. In an alternative embodiment directed to determining thepresence or absence of antibodies to H. capsulatum in a mammal, themethod comprises obtaining an antibody-containing sample from themammal, contacting the sample with a protein antigen of H. capsulatumwhich is bound by antibodies to H. capsulatum but which is notsubstantially bound by antibodies to each of Coccidioides immitis,Blastomyces dermatitidis or Candida sp., and determining whether anantibody in the sample immunoreacts with the protein antigen of H.capsulatum. In an additional embodiment for determining antibodies to H.capsulatum, the method comprises obtaining an antibody-containing samplefrom the mammal, contacting the sample with a protein antigen having anamino acid sequence as set forth in SEQ ID NO: 3, and determiningwhether an antibody in the sample immunoreacts with the protein antigen.

The invention is further directed to a method for determining thepresence or absence of a target-fungus protein antigen in a sample. Themethod generally comprises obtaining a sample to be tested for thepresence or absence of the target-fungus protein antigen, contacting thesample with an antibody or antibody fragment which is immunoreactivewith a target-fungus protein antigen, and determining whether theantibody or antibody fragment immunoreacts with the target-fungusprotein antigen. As directed to determining the presence or absence of aH. capsulatum protein antigen in a mammal, the method comprisesobtaining a sample to be tested for the presence or absence of the H.capsulatum protein antigen, contacting the sample with an antibody orantibody fragment which is immunoreactive with an antigen of H.capsulatum, but which is not substantially immunoreactive with antigensof each of Coccidioides immitis, Blastomyces dermatitidis or Candidasp., and determining whether the antibody or antibody fragmentimmunoreacts with the H. capsulatum protein antigen. In an alternativemethod for determining the presence or absence of a H. capsulatumprotein antigen in a mammal, the method comprises obtaining a sample tobe tested for the presence or absence of the H. capsulatum proteinantigen, contacting the sample with an antibody or antibody fragmentwhich is immunoreactive with a protein antigen having an amino acidsequence as set forth in SEQ ID NO: 3, and determining whether theantibody or antibody fragment immunoreacts with the protein antigen.

The invention is additionally directed to a kit that includes a reagentselected from, in one embodiment, one or more of the following: (i) atarget-fungus protein antigen identified according to the aforementionedmethod, (ii) a fragment of a target-fungus protein antigen identifiedaccording to the aforementioned method wherein the fragment is bound byantibodies to the target fungus but is not substantially bound byantibodies to the nontarget fungus, and (iii) a target-fungus antibodyor antibody fragment which immunoreacts with a target-fungus proteinantigen identified according to the aforementiond method. In analternative embodiment, the reagent is selected from one or more of thefollowing: (i) a protein antigen of H. capsulatum which is bound by H.capsulatum antibodies but which is not substantially bound by antibodiesto each of Coccidioides immitis, Blastomyces dermatitidis or Candidasp., (ii) a fragment of a H. capsulatum protein antigen wherein thefragment is bound by antibodies to H. capsulatum but is notsubstantially bound by antibodies to each of Coccidioides immitis,Blastomyces dermatitidis or Candida sp., and (iii) an antibody orantibody fragment which is immunoreactive with a H. capsulatum proteinantigen but which is not substantially immunoreactive with antigens ofeach of Coccidioides immitis, Blastomyces dermatitidis or Candida sp. Inyet another embodiment, the reagent is selected from: (i) a proteinantigen having an amino acid sequence as set forth in SEQ ID NO:3, (ii)a protein antigen that includes a portion of the amino acid sequence asset forth in SEQ ID NO:3 wherein the included portion is bound byantibodies to H. capsulatum but is not substantially bound by antibodiesto each of Coccidioides immitis, Blastomyces dermatitidis or Candidasp., (iii) an antibody or antibody fragment which is immunoreactive witha protein antigen having the amino acid sequence set forth in SEQ ID NO:3, and (iv) an antibody or antibody fragment which is immunoreactivewith a protein antigen that includes a portion of the amino acidsequence set forth in SEQ ID NO: 3 wherein the included portion is boundby antibodies to H. capsulatum but is not substantially bound byantibodies to each of Coccidioides immitis, Blastomyces dermatitidis orCandida sp. The kit also includes instructions for directing the use ofthe reagent for determining the presence or absence of the target fungusin a sample. In one embodiment, the instructions direct the use of thereagent for determining whether a mammal is presently infected or hasbeen previously infected with the target fungus. In another embodimentin which the reagent is an antibody, the instructions direct the use ofthe antibody reagent for determining the presence or absence of anantigen in a nonvertebrate sample or environment, such as a plant, food,feed, feed component, air, water, or other fluid sample.

Other features, objects and advantages of the present invention will bein part apparent to those skilled in the art and in part pointed outhereinafter. All references cited in the instant specification areincorporated by reference. Moreover, as the patent and non-patentliterature relating to the subject matter disclosed and/or claimedherein is substantial, many relevant references are available to askilled aritsan that will provide further instruction with respect tosuch subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the deduced amino acid sequence encoded byGH17 with threonine-rich regions of: (A) cellulase from the thermophilicbacterium Caldocellum saccharolyticum (SEQ ID NO: 4); (B), Leishimaniasurface antigen (SEQ ID NO: 5); and (C), integumentary mucin fromXenopus laevis (SEQ ID NO: 6) using the NCBI BLAST analysis program.Identical residues are indicated with a (|), and conserved residues aremarked with a (·)

FIG. 2 shows a hydropathy plot of the protein encoded by GH17.Hydropathy analysis was performed by the method of Hopp and Woods.Hydropathy values were averaged for a window of six amino acid residues.Positive numbers indicate hydrophilicity. The point of highesthydrophilicity (Average hydrophilicity=2.08, between residues 155-160)is marked with a broken vertical line.

FIG. 3 shows a Southern blot of genomic DNA of H. capsulatum probed withlabeled cDNA insert from clone GH17. Genomic DNA was digested with EcoRI(lane 1), PstI (lane 2), and SacI (lane 3), electrophoresed on a 1%agarose gel, and transferred to nylon membrane. The membrane was probedwith peroxidase-labeled CDNA insert of GH17 and washed underhigh-stringency conditions.

FIG. 4 shows the results of an immunoblot analysis of theimmunoreactivity and specificity of the β-galactosidase fusion proteinof the recombinant clone GH17. The immunoreactive fusion protein band isindicated by an arrow.

FIG. 4A shows a representative immunoblot demonstrating theimmunoreactivity of the fusion protein. Bacterial cell-lysates fromcells infected with GH17 were separated by SDS-PAGE andelectrophoretically transferred to nitrocellulose paper. The blot wasdeveloped with individual sera from patients with histoplasmosis (Lanes1-18). 16 of 18 sera had strong antibody reactivity with the fusionprotein, and two sera were weakly reactive (Lanes 6 and 9). Lane 19 wasdeveloped with a murine monoclonal antibody to β-galactosidase.

FIG. 4B demonstrates the antigenic specificity of recombinant H.capsulatum clone GH17β-galactosidase fusion protein by immunoblotanalysis. Lanes in various panels were developed as follows: Panel A,with sera from dogs infected with B. dermatitidis (n=6); B, with humansera from patients infected with B. dermatitidis (n=5); C, with humansera from patients infected with Candida albicans (n=5); D, with humansera from patients infected with Coccidioides immitis (n=12); E, withhuman histoplasmosis serum pool and anti-β-galactosidase antibody,respectively.

FIG. 5 shows an SDS-PAGE and immunoblot analysis of expression ofGH17-his in pPROEX-1™ protein expression vector.

In FIG. 5A, 10% SDS-PAGE was loaded with E. coli extract without IPTGinduction (lane 1), and after IPTG induction for 3 hr (lane 2). Theimmunoblots were developed with human histoplasmosis serum pool (1:500),enzyme-labeled anti-human IgG secondary antibody, and substrate.

FIG. 5B shows immunoblot analysis of eluted fractions of GH17-his fusionprotein separated by preparative SDS-PAGE in the model 491 BioRad PrepCell. Aliquots (10 μl) from the Prep Cell fractions were separated bySDS-PAGE on 12% gels, immunoblotted, and developed as described for FIG.5a. Lane 1, IPTG induced E. coli extract (2 μl); Lanes 2-4 containpositive fractions from the Prep Cell.

FIG. 6 shows an immunoblot analysis of H. capsulatum yeast antigenextract developed with: lane 1, mouse antibody to GH17-his fusionprotein (1:500); lane 2, normal mouse serum (1:500); and lane 3, mouseantibodies to H. capsulatum yeast extract (1:500).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed toward protein antigens which arespecific to a target fungus, to methods of obtaining such antigens, toantibodies to such antigens, and to assays employing such antigens andantibodies.

An antigen specific to a particular target fungus is identified by a)obtaining a cDNA gene expression library for the target fungus, b)expressing target fungal proteins from the cDNA gene expression library,c) obtaining antisera to the target fungus which has antibodies to thetarget fungus, d) obtaining antisera to the nontarget fungus which hasantibodies to the nontarget fungus and e) identifying a target-fungusprotein which is bound by the antibodies to the target fungus, but whichis not substantially bound by the antibodies to the nontarget fungus.

The steps of the above method need not be performed in any particularorder. In a preferred embodiment of the above method, the cDNAexpression library is first screened with an antisera to the targetfungus. Clones which are highly reactive to the target fungus are thusidentified, and these selected clones are then screened with antisera tothe nontarget fungus. The clones which produce a protein which is boundby antibodies in the antisera to the target fungus but not by antibodiesin the antisera to other microorganisms (e.g. nontarget fungi) are thenidentified. Various manipulations known in the art can utilize theseclones to produce and substantially purify the protein antigens encodedby the clones. The protein antigens can then be used as discussed below.

The procedures disclosed herein which involve the molecular manipulationof nucleic acids are known to those skilled in the art. See generallyFredrick M. Ausubel et al. (1995), “Short Protocols in MolecularBiology”, John Wiley and Sons, and Joseph Sambrook et al. (1989),“Molecular Cloning, A Laboratory Manual”, second ed., Cold Spring HarborLaboratory Press.

A used herein, the term “target fungus” (pl. “target fungi”) denotes anyfungus for which there is a particular interest. Examples of targetfungi are fungi which are pathogens of animals or plants, allergens,food spoiling agents, or food sources. Preferred target fungi includeAscomycetes. More preferred target fungi are those fungi in the genusGymnoascaceae. Even more preferred fungi are those in the genusAjellomyces, and the most preferred target fungus is H. capsulatum. A“nontarget fungus” may be any fungus that is desired to be distinguishedfrom the target fungus, for identification or diagnostic purposes, by animmunoassay. In general, the nontarget fungus includes at least oneantigenic determinant in common with the target fungus. As such, thenontarget fungus can cross react with an antibody that recognizes thisshared antigenic determinant. Exemplary nontarget fungi may be selectedfrom fungi known to cause a disease with similar symptoms as the targetfungus.

The cDNA gene expression library of the target fungus may be obtainedfrom a commercial source or prepared by methods known in the art, usingan appropriate morphological stage of the target fungus. An easilyculturable stage such as mycelium is preferred. Common vectors used forthis purpose are plasmid, bacteriophage, and mammalian cells. Apreferred expression library is one utilizing a phage such asbacteriophage λgt11, with poly(A)+mRNA.

The target fungus proteins are expressed by any method known in the art.For the preferred λgt11 expression library, the proteins are generallyexpressed by induction with IPTG.

Antisera to the target fungus may be obtained in any manner known in theart. Preferably such antisera are obtained by preparing an antigen whichwill, upon immunization of an animal with the fungus, yield an antiserawhich contains a population of antibodies which, in sum, willimmunoreact to many antigens of the target fungus. An unfractionatedpreparation of the target fungus is thus a preferred antigenpreparation. However, a fractionated preparation could also be employed.When the target fungus is pathogenic to vertebrates which are capable ofmounting an immunological response to the fungus, the preferredscreening antisera is serum from infected individuals. A more preferredscreening antisera is pooled serum from several infected individuals.Antisera to the nontarget fungus can be obtained by methods similar tothe methods used to prepare antisera to the target fungus.

Target-fungal proteins which are bound by the target-fungus antibodiesbut which are not substantially bound by the nontarget-fungus antibodiescan be identified by screening each of the antisera by appropriateimmunological assays known in the art. The terms “immunoreact”, “bind”,“are bound to” or grammatical derivations thereof are usedinterchangeably herein, and refer to the capability of an antibody or anantibody fragment to specifically attach to an antigen at the antibody'sFab binding site. Exemplary screening assays include precipitin assaysand label-based assays. Preferably, the antigen is identified from theexpressed cDNA gene expression library by blotting the expressedproteins (e.g. from phage plaques) onto membranes then screening themembranes with the antisera made to each fungus.

The degree of immunological cross-reactivity for the identifiedtarget-fungus protein (generally referred to herein as a target-fungusspecific antigen) is preferably sufficiently low so that tests thatdetect the antigen or antibodies to the antigen are useful for reliablydistinguishing the fungus from nontarget fungi. The degree ofimmunological cross-reactivity with nontarget fungi and/or othermicroorganisms of interest, when assessed by immunoassay (for exampleWestern blot or ELISA), is preferably less than about 10%, morepreferably less than about 5%, still more preferably less than about 2%,even more preferably less than about 1%, and most preferably less thanabout 0.1%.

The identified target-fungus specific protein antigen is morespecifically characterized as follows, with the various aspects definingthe protein to be considered both independently and in combination. Thetarget fungus-specific protein antigen is preferably substantially freefrom non-protein determinants such as carbohydrates, phosphorylcholine,and/or other moieties which, when attached to or otherwise associatedwith the protein, would reduce the immunological specificity of theprotein.

The term “specific” is used herein to denote an antigen which is notpresent in nontarget fungi. When referring to an antibody or to anassay, “specific” denotes a substantial lack of cross-reactivity withnontarget fungi. As an example, an antigen of a target fungus whichinfects mammals would be specific if antibodies in serum produced by atarget fungus-infected vertebrate bound to the antigen, but seraproduced in vertebrates which are infected with a nontarget fungus donot substantially cross-react with the antigen. The extent ofcross-reactivity can be more specifically characterized with regard to aset (i.e. group or population) of samples being evaluated. In a samplepopulation known to comprise the target-fungus antigen or antibodies tothe antigen, the presence of the antigen or antibody is correctlydetermined in preferably at least about 90% of the samples, morepreferably in at least about 95% of the samples and most preferably inat least about 99% of the samples. Conversely, in a sample populationknown to both (i) lack the target fungus antigens or antibodies theretoand (ii) comprise a nontarget fungus antigen or antibodies thereto, theabsence of the target fungus antigen or antibody thereto is correctlydetermined in preferably at least about 90% of the samples, morepreferably in at least about 95% of the samples and most preferably inat least about 99% of the samples. As another example, an antigen on aplant pathogenic fungus is specific if antisera made to that antigendoes not substantially cross-react with antigens on other nontargetfungi which might be present in the same environment as the plantpathogenic fungus.

Target-fungus specific protein antigens with such cross-reactivity canbe varied from the identified protein antigen, but will preferably havean amino acid sequence which has a sequence identity or, alternatively,a homology of at least about 75%, more preferably at least about 90%,even more preferably at least about 95% and most preferably at leastabout 98% relative to the amino acid sequence of the identified proteinantigen or, alternatively, as encoded by the cDNA clone thereof.

In a preferred embodiment the target fungus is H. capsulatum. Preferrednontarget fungi are C. immitis, B. dermatitidis, and Candida sp. H.capsulatum proteins expressed from a cDNA expression library arescreened, preferably by immunoblot, against antisera to H. capsulatumand at least one of the nontarget fungi. The antisera are preferablyprovided by pooled sera from individuals infected with the target andnontarget fungi. Where the target fungus is H. capsulatum, a preferredprotein antigen has the amino acid sequence set forth as SEQ ID NO:3.(See example). In another embodiment, the protein is a H.capsulatum-specific protein antigen and has an amino acid sequence whichincludes at least a portion of the amino acid sequence set forth as SEQID NO:3 which is specific for H. capsulatum, the included portion being,in a preferred protein, at least 5 amino acids in length. The H.capsulatum-specific antigen identified in this manner is notsubstantially cross-reactive with antisera to the nontarget fungus. (SeeExample).

The target-fungus specific antigen, identified for example by theafore-described screening protocols, can be isolated and produced insubstantially purified form according to methods known in the art.Briefly, the cDNA clone corresponding to the identified target-fungusspecific antigen, or more generally any nucleic acid polymer encodingthe target-fungus specific protein antigen, can be incorporated into anexpression vector for recombinant production of the protein antigen, asdiscussed below.

The nucleic acid polymer can have the cDNA nucleotide sequence of theisolated cDNA clone. The nucleic acid polymer can, alternatively, have amRNA nucleotide sequence corresponding to the cDNA sequence. Where thetarget fungus is H. capsulatum, the nucleic acid polymer preferably hasthe cDNA nucleotide sequence set forth as SEQ ID NO:2 or a mRNAnucleotide sequence corresponding to the sequence set forth as SEQ IDNO:2.

In an additional embodiment, the nucleic acid polymer can encode afungus-specific protein antigen and have an nucleotide sequence whichincludes at least a portion of the nucleotide sequence of the isolatedcDNA, the included portion being at least 15 base pairs in length. In afurther embodiment, the nucleic acid polymer can be at least 15 basepairs in length and encode a fungus-specific protein antigen having anamino acid sequence which has a sequence identity or, alternatively, ahomology of at least about 75%, more preferably at least about 90%, evenmore preferably at least about 95% and most preferably at least about98% relative to the amino acid sequence encoded by the isolated cDNA.

The above-disclosed nucleic acid polymer which encodes a fungus-specificprotein antigen is preferably used to create a vector which is used, forexample, to replicate or translate the nucleic acid polymer. Translationof the nucleic acid polymer is preferably accomplished by an expressionvector by methods known in the art. The expression vector can be, forexample, a hybrid plasmid, a virus, or other nucleic-acid-polymerconstruct which is suitable for use in expressing the antigen in aeukaryotic or prokaryotic host-cell, in vitro, according to methodsknown in the art. In the case of H. capsulatum, preferred expressionvectors are λgt11 and the pProEXm-1 protein expression system, whichproduces a fusion protein containing 6 histidines.

A host cell can be transformed with the above-disclosed vector forrecombinant production of the target fungus-specific protein antigen.The host cell can be, for example, a bacterial host cell such as E.coli, a yeast cell, a mammalian cell, or any other suitable host cell inwhich the antigen can be expressed and from which the antigen can besubstantially isolated and purified.

The isolated fungus-specific protein antigen can be utilized to producean antibody specific for the antigen. The antigenic protein or fragmentagainst which the antibody is raised and to which the antibody binds ispreferably substantially purified, and is further characterized as setforth above, with the various aspects defining the protein antigen to beconsidered both independently and in combination.

The antibody may be a mono-specific antibody. The monospecific antibodymay be a monoclonal antibody produced, for example, by the method ofGalfre et al., Nature 266:550 (1977). Alternatively, the monospecificantibody may be a recombinant antibody produced, for example, by themethod of Lowman et al, Biochemistry 30:10832-10838 (1991).

The antibody can also be a polyclonal antibody. The polyclonal antibodycan be prepared by immunizing a mammal such as a mouse or rabbit withthe fungus-specific antigen and subsequently isolating the serumtherefrom to obtain an antiserum that contains the polyclonalantibodies. If the fungus is a pathogen of a vertebrate animal, such asH. capsulatum, polyclonal antibodies reactive to the specific antigenare generally produced in the serum of an infected animal. That serummay be collected and utilized as a polyclonal antiserum to the fungus.

The target fungus-specific antigen, and antibodies made to that specificantigen can be utilized in assays to determine the presence or absence,in a sample, of antigens or antibodies which are indicative of ordiagnostic for the target fungus.

Any portion of the antigen which is specific for the fungus may beutilized for identifying the target fungus in a sample, and specificpeptide sequences as small as five amino acids in length may be easilyobtained by methods known in the art. These specific fragments may, forexample, be used alone or they may be engineered by methods known in theart to be part of a fusion protein, preferably comprising two domains, afirst domain that includes at least a portion of the amino acid sequenceencoded by the nucleic acid polymer, the included portion being at least5 amino acids in length, and a second domain that includes the aminoacid sequence of another protein or polypeptide. In a preferredembodiment, the second domain includes the amino acid sequence of aprotein from the expression vector, such as β-galactosidase or otherprotein incorporated in an expression system, which may facilitateexpression and/or subsequent purification of the expressed antigen fromthe host-cell lysate.

In the case of a fungus disease of humans and other animals, the testswill preferably allow one to distinguish the fungal disease from otherclinical conditions, especially from other fungal infections. Withoutbeing bound to a particular theory not specifically required in theclaims, the target-fungus specific antigens result from theabove-disclosed method because the expressed proteins from the cDNAexpression library do not contain carbohydrate moieties which would bepresent in fungal antigen preparations prepared by prior art methods.Prior art methods of immunizing vertebrates with components of thetarget fungus generally failed to identify specific antigens because theimmune system of the immunized vertebrate would mount an immune responseto the antigenic carbohydrate moieties of the immunogen target fungus.However, the carbohydrate moieties of the immunogen target fungus areoften also present in the nontarget fungi, thus leading tocross-reactivities with the nontarget fungi. In particular, fungi whichare closely related taxonomically, such as H. capsulatum and B.dennatitidis, generally have more antigenic determinants in common thanfungi which are less closely related, such as H. capsulatum and Agaricusbisporus, the common cultivated mushroom. As such, the present method isparticularly suited for developing immunoassays for target fungi whichmight be confused for closely related nontarget fungi.

In the preferred embodiment, a test for histoplasmosis developed with anantigen specific for H. capsulatum (as in the Example) allows adetermination of the presence or absence of antibodies to H. capsulatumwhich do not immunoreact with nontarget fungi present in thesample—particularly Coccidioides immitis, Blastomyces dermatiditis,and/or Candida species. Thus, the invention as applied to H. capsulatumprovides diagnostic methods (i.e., assays) for determining whether amammal has been infected with H. capsulatum. These methods, combinedwith clinical observations and findings based on known and/or onlater-developed techniques, facilitate diagnosis of histoplasmosis.

One embodiment of the assay method as applied to a target fungus isreferred to herein as an antibody assay. This method comprises detectingthe presence or absence of antibodies to a target fungus-specificantigen in a sample obtained from a vertebrate. The presence or absenceof the target fungus-specific antibodies are detected by contacting thesample with an antigen specific to the target fungus and determiningwhether the sample contains antibodies that bind to the targetfungus-specific antigen. The preferred antigen is further characterizedas set forth above, with the various aspects defining the antigen to beconsidered both independently and in combination. Exemplary antibodyassays, discussed in more detail below, include precipitin-basedimmunoassays, indirect label-based immunoassays, direct label-basedimmunoassays and inhibition/competitive-type label-based immunoassays.The presence of target fungus-specific antibodies in a sample obtainedfrom the mammal is evidence of current and/or past exposure or infectionwith the target fungus. In an alternative embodiment, referred to hereinas an antigen assay, the method comprises detecting the presence orabsence of antigens specific to the target fungus in a sample. Thepresence or absence of target fungus-specific antigens is detected bycontacting the sample with an antibody capable of binding to a targetfungus-specific antigen and determining whether the antibody binds tothe target fungus-specific antigen. The preferred target fungus-specificantibody is as set forth above, with the various aspects defining theantibody to be considered both independently and in combination. Thetarget fungus-specific antigen being detected is further characterizedas set forth above, with the various aspects defining the antigen to beconsidered both independently and in combination. Exemplary antigenassays, discussed in more detail below, include precipitin-basedimmunoassays, indirect label-based immunoassays, direct label-basedimmunoassays and inhibition/competitive-type label-based immunoassays.The presence of antigens specific to the target fungus in a sample isevidence of the presence of the target fungus in the sample.

As applied to H. capsulatum, the detection of antibodies which bind tothe H. capsulatum-specific antigen in a sample from a mammal is evidenceof past or current infection with H. capsulatum. Similarly, thedetection of H. capsulatum-specific antigens in a sample from a mammalis strong evidence of current infection with H. capsulatum.

The following additional concerns are applicable to either of theaforementioned antibody assay or antigen assay as applied to a targetfungus such as H. capsulatum. The mammals from which a sample isobtained are preferably humans, domestic livestock and/or pets which aresuspected of being or known to be susceptible to fungal infection by thetarget fungus (e.g. H. capsulatum). The sample can be a blood sample, aplasma sample, a serum sample, a urine sample, a sputum sample, a salivasample or any other biological sample obtained from the mammal which issuspected of potentially having antibodies to the target fungus (e.g. H.capsulatum) or having target-fungus (e.g. H. capsulatum) antigens.

The sample can be pretreated prior to testing the sample in the assay.Exemplary pretreatment steps can include concentrating the sample and/oreliminating interfering substances (e.g. acid in urine or rheumatoidfactors in serum). Other pretreatment steps will be apparent to a personof skill in the art. Moreover, additionally or alternatively todetecting whether antigen-antibody binding occurs in either of theaforementioned general methods, the extent of such binding can bequantitatively determined using methods known in the art.

The antibody assays of the present invention can be more specificallycharacterized according to a variety of formats set forth below and/orknown in the art. One approach includes the use of precipitin-basedimmunoassays. For example, the presence or absence of the antibodies canbe detected by layering a first solution including the targetfungus-specific antigen over the undiluted (i.e., neat) sample or over asecond solution including the sample, the layered solution typicallybeing formed in a container such as a test-tube, and observing thelayered solution for the formation or the lack of formation of aprecipitate comprising bound antigen and antibody. The amount of targetfungus-specific antigen in the first solution is preferably an amountwhich is necessary, on average, to form a precipitate with samples drawnfrom a vertebrate known to contain antibodies to the target fungus. Thesteps of this approach can, alternatively, be repeated, in parallel orin series, using various amounts of the target fungus-specific antigenin the first solution, with the amount of antigen in the solutionvarying over a range which includes an amount which is about, onaverage, necessary to form a precipitate with samples drawn from mammalsknown to have histoplasmosis. Precipitin-based immunoassays can also becarried out in gels such as agar or polyacrylamide gels or theirequivalents known in the art, by methods typically referred to asimmunodiffusion, immunoelectrophoresis, counterimmunoelectrophoresisand/or rocket electrophoresis, among others. For example, the presenceor absence of the antibodies are detected by placing a first solutionincluding the target fungus-specific antigen in a gel or in a welladjacent to a gel, placing the undiluted sample or a second solutionincluding the sample, in a gel or in a well adjacent to a gel, allowingthe target fungus-specific antigen and antibodies to diffuse in the gel,and observing the gel for the formation or the lack of formation of aprecipitate comprising bound antigen and antibody. Turbidometric ornephelometric-type assay formats can also be employed. Precipitin-basedimmunoassays offer the advantage of not requiring a solid-phase matrix,and as such, may be suited for particular applications known in the art(e.g. automated systems).

Another approach for the antibody assay of the present inventionincludes the use of label-based assay techniques, including direct,indirect and/or inhibition/competitive radioimmunoassays, enzyme-linkedimmunoabsorbant assays (ELISA), immunofluorescent assays,immunochromatographic assays, and other techniques known in the art. Forexample, the presence or absence of the antibodies can be detected usingan indirect label-based immunoassay by immobilizing the targetfungus-specific antigen on a solid-phase, contacting the immobilizedantigen with the undiluted sample or with a solution including thesample to allow any target fungus-specific antibody which may be presentin the sample to specifically bind to the immobilized antigen, therebyforming a first immobilized complex which includes eithersolid-phase/target fungus-specific antigen or solid-phase/targetfungus-specific antigen/antibody depending on whether the targetfungus-specific antibody was present in the sample, washing the firstimmobilized complex to remove any unbound target fungus antibody and/orother serum components, contacting the first immobilized complex with adetectable secondary antibody capable of binding to the targetfungus-specific antibody, thereby forming a second immobilized complexwhich includes either solid-phase/target fungus-specific antigen orsolid-phase/target fungus-specific antigen/antibody/secondary-antibodydepending on whether target fungus antibody was present in the sample,washing the second immobilized complex to remove any unbound secondaryantibody, and detecting the presence or absence of the secondaryantibody on the second immobilized complex. The detectable secondaryantibody can be labeled according to methods known in the art or laterdeveloped. For example, the secondary antibody can be a radiolabeledantibody (e.g. an antibody labeled with a gamma-emitting ¹²⁵¹I isotope)which is detected by radiographic methods or with instruments such ascounters which measure the level of radioactivity present. The secondaryantibody can also be an enzyme-conjugated antibody (e.g. an antibodyconjugated with alkaline phosphatase, horseradish peroxidase, or otherenzyme) which is detected by contacting the enzyme-conjugated antibodywith a color-producing enzyme substrate. The secondary antibody canalternatively be tagged with biotin (or an equivalent) or with afluorochrome (e.g. fluorescein and rhodamine) or a dye or other coloredsubstance (e.g. colloidal gold) which can be detected visually or byknown spectroscopic methods.

In another example of an indirect label-based immunoassay, the presenceor absence of target fungus-specific antibodies can be detected using aWestern blot format. This method is particularly advantageous in that itincludes a step for separating the target fungus-specific antigen fromother proteins in the sample in which it is present (e.g. for isolatingrecombinantly-produced target fungus-specific antigen present in ahost-cell lysate). This method includes electrophoretically separating atarget fungus-specific protein antigen electrophoretically, transferringthe separated protein antigen to a solid-phase membrane (e.g. anitrocellulose or nylon membrane), contacting the solid-phase/targetfungus-specific antigen complex with an undiluted sample or with asolution including the sample to allow any target fungus-specificantibody which may be present in the sample to bind to the antigen, andto form a first complex which includes either solid-phase/targetfungus-specific antigen or solid-phase/target fungus-specificantigen/antibody, depending on whether antibody was present in thesample, washing the first complex to remove any unbound antibody,contacting the first complex with a detectable secondary antibodycapable of binding to the solid phase-bound antibody, thereby forming asecond immobilized complex which includes either solid-phase/targetfungus-specific antigen or solid-phase/target fungus-specificantigen/antibody/secondary-antibody depending on whether targetfungus-specific antibody was present in the sample, washing the secondimmobilized complex to remove any unbound secondary antibody, anddetecting the presence or absence of the secondary antibody on thesecond immobilized complex. The detectable secondary antibody can, forexample, be radiolabeled, enzyme- conjugated, tagged with afluorochrome, or dyed as described above.

The presence or absence of target fungus-specific antibodies can, inanother exemplary method, be detected using a direct label-basedimmunoassay. This method includes immobilizing a firstanti-immunoglobulin antibody (e.g. IgG) capable of binding to the targetfungus antibody being detected on a solid-phase (e.g. beads, membrane,matrix, etc.), contacting the immobilized anti-immunoglobulin antibodywith an undiluted sample or with a solution including the sample to forma first complex which includes eithersolid-phase/anti-immunoglobulin-antibody orsolid-phase/anti-immunoglobulin-antibody/target fungus-specificantibody, depending on whether target fungus-specific antibody waspresent in the sample, washing the first complex, contacting the firstcomplex with a labeled target fungus-specific antigen to allow anytarget fungus-specific antibody present in the first complex to bind tothe target fungus-specific antigen and to form a second complexcomprising either solid-phase/anti-immunoglobulin-antibody orsolid-phase/anti-immunoglobulin-antibody/target fungus-specificantibody/target fungus-specific antigen, depending on whether targetfungus-specific antibody was present in the sample, washing the secondcomplex to remove any unbound labeled target fungus-specific antigen,and detecting whether the labeled target fungus-specific antigen ispresent or absent in the second complex. The target fungus-specificantigen can be labeled according to methods now known in the art orlater developed, including, for example, being radiolabeled,enzyme-conjugated, tagged with a fluorochrome, dyed or otherwiseassociated with a colored material, as described above.

The presence or absence of target fungus-specific antibodies can, in afurther exemplary method, be detected using a inhibition/competitivelabel-based immunoassays. This method includes establishing a baselinereading for a control assay by immobilizing a target fungus-specificantigen on a solid-phase, contacting the immobilized targetfungus-specific antigen with a detectable (e.g. labeled) targetfungus-specific antibody to form a control complex includingsolid-phase/target fungus-specific-antibody/detectable targetfungus-specific antibody, washing the control complex to remove anyunbound detectable target fungus-specific antibody therefrom, anddetecting the baseline level of target fungus-specific antibody bound tothe immobilized target fungus-specific antigen on the control complex.The method further includes, in a separate, independent test assay,immobilizing a target fungus-specific antigen on a solid-phase,contacting the immobilized target fungus-specific antigen with both (1)a detectable (e.g. labeled) target fungus-specific antibody and (2) anundiluted sample or with a solution including the sample to allow anytarget fungus-specific antibody which may be present in the sample tobind to at least some of the immobilized target fungus-specific antigenand to thereby form a test complex in which at least some of the bounddetectable target fungus-specific antibody may have been competitivelyinhibited from binding to the immobilized target fungus-specificantigen, depending on whether target fungus-specific antibody waspresent in the sample, washing the test complex to remove any unboundtarget fungus-specific antibody, detecting the level ofdetectable-target fungus-specific antibody bound to the test complex,and comparing the level of detectable-target fungus-specific antibodybound to the test complex to the baseline level of detectable targetfungus-specific antibody bound to the control complex, a decrease insuch level indicating the presence of target fungus-specific antibody inthe sample. The detectable target fungus-specific antibody can, forexample, be radiolabeled, enzyme-conjugated, tagged with a fluorochrome,dyed or otherwise associated with a colored material as described above.

The approaches set forth above for determining the presence or absenceof antibodies to target fungus-specific antibody in a sample are to beconsidered exemplary and non-limiting of the many formats known in theart by which a sample suspected of including antibodies is contactedwith an antigen and the presence or absence and/or quantitative extentof antigen-antibody binding is determined. Moreover, the exact sequenceof steps is not narrowly critical and can be varied as is appropriate inthe art. Certain steps may be omitted altogether and/or combined withother steps. For example, the sample and-labeled antigen can, in someassay formats, be added together. As another example, assays may notrequire a washing step to remove unbound antibodies. Assays such asimmunochromatographic assays where reactants flow across and/or throughthe solid phase are exemplary. Certain additional steps may also beadded, in series and/or in parallel combination, to the aforementionedsteps, as appropriate in the art. For example, the assays can optionallyinclude one or more blocking steps or proteins or detergents in thediluent to decrease the non-specific binding of antibodies, primary orsecondary, to the solid-phase. The assays of the invention can also beautomated, with appropriate modifications to the described steps. All ofthe above antibody assays are effective in detecting targetfungus-specific antibodies for any fungus, including H. capsulatum,which is capable of eliciting an antibody response in a vertebrate.

The antigen assays of the present invention are useful in any situationwhere a determination of the presence or absence of a target fungus isdesired. Examples include the determination of the presence of a targetfungus in an animal or plant suspected of being infected with the targetfungus, in a food or feed suspected of being contaminated with thetarget fungus, in inorganic materials such as soil or air for thedetermination of the presence of an allergenic or pathogenic targetfungus, and for the identification of a specific target fungus where theidentity of the fungus is unknown.

The antigen assays of the present invention can be more specificallycharacterized according to a variety of formats set forth below and/orknown in the art. One approach includes the use of precipitin-basedimmunoassays. For example, the presence or absence of the targetfungus-specific antigens in the sample can be detected by layering afirst solution including the target fungus-specific antibody over theundiluted sample or over a second solution including the sample, thelayered solution typically being formed in a container such as atest-tube, and observing the layered solution for the formation or thelack of formation of a precipitate comprising bound antigen andantibody. The amount of target fungus-specific antibody in the firstsolution is preferably an amount which is necessary, on average, to forma precipitate with samples previously known to comprise the targetfungus. The steps of this approach can, alternatively, be repeated, inparallel or in series, using various amounts of the targetfungus-specific antibody in the first solution, with the amount ofantibody in the solution varying over a range which includes an amountwhich is about, on average, necessary to form a precipitate with samplespreviously known to contain the target fungus-specific antigen.Precipitin-based immunoassays can also be carried out in gels such asagar or polyacrylamide gels or their equivalents known in the art, bymethods typically referred to as immunodiffusion, immunoelectrophoresis,counterimmunoelectrophoresis and/or rocket electrophoresis, amongothers. For example, the presence or absence of the antigens aredetected by placing a first solution including the targetfungus-specific antibody in a gel or in a well adjacent to a gel,placing the undiluted sample or a second solution including the sample,in a gel or in a well adjacent to a gel, allowing the targetfungus-specific antibody and antigens to diffuse within the gel andobserving the gel for the formation or the lack of formation of aprecipitate comprising bound antigen and antibody.

Another approach for the antigen assay of the present invention includesthe use of label-based assay techniques, including direct, indirectand/or inhibition/competitive radioimmunoassays, enzyme-linkedimmunoabsorbant assays (ELISA), immunofluorescent assays,immunochromatographic assays, and other techniques known in the art. Forexample, the presence or absence of the antigens can be detected in adirect sandwich-type format by immobilizing target fungus-specificantibody on a solid-phase, contacting the immobilized antibody with anundiluted sample or with a solution including the sample to allow anytarget fungus-specific antigen which may be present in the sample tobind to the immobilized antibody, thereby forming a first immobilizedcomplex which includes either solid-phase/target fungus-specificantibody or solid-phase/target fungus-specific antibody/targetfungus-specific antigen, depending on whether the target fungus-specificantigen was present in the sample, washing the first immobilized complexto remove any unbound target fungus-specific antigen, contacting thecomplex with a detectable secondary antibody capable of binding to adifferent epitope on the target fungus-specific antigen, thereby forminga second immobilized complex which includes either solid-phase/targetfungus-specific antibody or solid-phase/target fungus-specificantibody/target fungus-specific antigen/secondary-antibody, depending onwhether target fungus-specific antigen was present in the sample,washing the second immobilized complex to remove any unbound secondaryantibody, and detecting the presence or absence of the secondaryantibody on the second immobilized complex. The secondary antibody can,for example, be radiolabeled, enzyme-conjugated, tagged with afluorochrome or dyed as described above.

In another example of an indirect label-based immunoassay, the presenceor absence of target fungus-specific antigen can be detected using aWestern blot format. This method includes electrophoretically separatingproteins contained in the sample in a gel (e.g. such as a polyacrylamidegel), electrophoretically transferring the separated proteins to asolid-phase membrane (e.g. a nitrocellulose membrane), contacting theseparated proteins with an unlabeled target fungus-specific antibody toallow any target fungus-specific antigen which may have been present inthe sample to bind to the antibody and form a first complex withincludes either solid-phase/target fungus-specific antigen orsolid-phase/target fungus-specific antigen/target fungus-specificantibody, depending on whether target fungus-specific antigen waspresent in the sample, washing the first complex to remove unboundtarget fungus-specific antibody, contacting the first complex with adetectable secondary antibody to form second complex which includeseither solid-phase/target fungus-specific antigen or solid-phase/targetfungus-specific antigen/target fungus-specificantibody/secondary-antibody, depending on whether target fungus-specificantigen was present in the sample, and detecting the presence or absenceof the secondary antibody bound to the antigen in the second complex.The secondary antibody can, for example, be radiolabeled,enzyme-conjugated, tagged with biotin or an equivalent thereto, afluorochrome, dyed or otherwise associated with a colored material asdescribed above.

In an exemplary direct Western blot immunoassay, the presence or absenceof the antigens in the sample can be detected using a method whichincludes electrophoretically separating proteins contained in the samplein a gel (e.g. such as a polyacrylamide gel), transferring the separatedproteins to a solid-phase membrane (e.g. a nitrocellulose or nylonmembrane), contacting the transferred proteins with a detectable targetfungus-specific antibody to allow any target fungus-specific antigenwhich may have been present in the sample to bind to the targetfungus-specific antibody and form a complex which includes eithergel/target fungus-specific antigen or gel/target fungus-specificantigen/target fungus-specific antibody, washing any unbound targetfungus-specific antibody away from the gel and detecting the presence orabsence of labeled target fungus-specific antibody bound to the antigenin the gel. The target fungus-specific antibody can, for example, beradiolabeled, enzyme-conjugated, tagged with a fluorochrome or dyed, asdescribed above.

Alternatively, the presence or absence of the antigens in a sample canbe detected in an inhibition/competitive-type format by mixing asolution including a detectable (e.g. labeled) target fungus-specificantibody with the undiluted sample or with a solution including thesample to allow any target fungus-specific antigen which may be presentin the sample to bind with the detectable target fungus-specificantibody and to form a test solution which includes either unbounddetectable target fungus-specific antibody or a detectable targetfungus-specific antibody/target fungus-specific antigen complexdepending on whether target fungus-specific antigen was present in thesample. The method further includes immobilizing a targetfungus-specific antigen on a solid-phase, contacting the immobilizedtarget fungus-specific antigen with the test solution to allow anyunbound detectable target fungus-specific antibody present in the testsolution to bind with the immobilized target fungus-specific antigen andform an immobilized complex including solid-phase/target fungus-specificantigen or solid-phase/target fungus-specific antigen/detectable targetfungus-specific antibody depending on whether target fungus-specificantigen was present in the sample, washing the solid-phase to remove anyunbound target fungus-specific antibody, and measuring the presence orabsence of the detectable target fungus-specific antibody on theimmobilized complex. The detectable antibody can be radiolabeled,enzyme-conjugated, tagged with a fluorochrome or dyed, as describedabove. In an alternative variation on this type of format, an antigenassay can include immobilizing a target fungus-specific antibody on asolid-phase, contacting the immobilized antibody with both (1) adetectable (e.g. labeled) target fungus-specific antigen and (2) anundiluted sample or a solution including the sample to allow any targetfungus-specific antigen which may be present in the sample to bind to atleast some of the immobilized target fungus-specific antibody and tothereby form a test complex in which at least some of the detectabletarget fungus-specific antigen may have been competitively inhibitedfrom binding to the immobilized target fungus-specific antibody,depending on whether target fungus-specific antigen was present in thesample, washing the test complex to remove any unbound targetfungus-specific antigen, and detecting the level of detectable targetfungus-specific antigen bound to the test complex. If desired, the levelof detectable target fungus-specific antigen bound to the test complexcan be compared to a baseline level of detectable target fungus-specificantigen bound to a control complex, with a decrease in such levelindicating the presence of target fungus-specific antigen in the sample.The detectable target fungus-specific antigen can, for example, beradiolabeled, enzyme-conjugated, tagged with biotin or an equivalentthereto, a fluorochrome, dyed or otherwise associated with a coloredmaterial as described above.

The approaches set forth above for determining the presence or absenceof target fungus-specific antigens in a sample are to be consideredexemplary and non-limiting of the many formats known in the art by whicha sample suspected of including antigens is contacted with an antibodyand the presence or absence and/or quantitative extent ofantigen-antibody binding is determined. Moreover, the exact sequence ofsteps is not narrowly critical and can be varied as is appropriate inthe art. Certain steps may be omitted altogether and/or combined withother steps. For example, the sample and labeled antigen can, in someassay formats, be added together. As another example, assays may notrequire a washing step to remove unbound antibodies. Assays such asimmunochromatographic assays where reactants flow across and/or throughthe solid phase are exemplary. Certain additional steps may also beadded, in series and/or in parallel combination, to the aforementionedsteps, as appropriate in the art. For example, the assays can optionallyinclude one or more blocking steps to decrease the non-specific bindingof antibodies, primary or secondary, to the solid-phase. The assays ofthe invention can also be automated, with appropriate modifications tothe described steps. All of the above antigen assays are effective indetecting target fungus-specific antigens for any fungus, including H.capsulatum.

Kits are provided which are suitable for use in performing theaforementioned assay methods to facilitate diagnosis of histoplasmosisin humans and other mammals. In one embodiment, an assay kit of thepresent invention can include labeled and/or unlabeled targetfungus-specific antigen, as described above, in quantities sufficient tocarry out the assays of the present invention. In another embodiment, anassay kit can include labeled and/or unlabeled antibodies to an targetfungus-specific antigen, as described above, in quantities sufficient tocarry out the assays of the present invention. In a further embodiment,an assay kit of the present invention can include both labeled and/orunlabeled target fungus-specific antigen and labeled and/or unlabeledantibody thereto, each as described above, in quantities sufficient tocarry out the assays of the present invention. In any of theaforementioned embodiments, an assay kit can also further comprise knownpositive and/or negative control samples, other reagents useful incarrying out the assays of the present invention (e.g. radiolabeledsecondary antibodies and/or enzyme-conjugated secondary antibodies alongwith the corresponding color-producing enzyme substrate therefore), andinstructions for carrying out the assay methods. Kits as provided abovemay be utilized for any target fungus, including H. capsulatum.

The following example illustrates the invention, but is not to be takenas limiting the various aspects of the invention so illustrated.

EXAMPLE MOLECULAR CLONING AND CHARACTERIZATION OF A RECOMBINANTHISTOPLASMA CAPSULATUM ANTIGEN FOR ANTIBODY DIAGNOSIS OF HUMANHISTOPLASMOSIS

MATERIALS AND METHODS

Fungi and culture conditions. H. capsulatum G217B, a North Americanisolate, was obtained from the American Type Culture Collection (ATCC#26032, Rockville, Md.). Mycelial-phase organisms were cultured in ashaking water bath at 25° C. in broth containing 2% glucose and 1% yeastextract. Yeast-phase organisms were grown at 37° C. in HMM broth(Gibco-BRL, Gaithersburg, Md.) supplemented with 18.2 g of glucose, 1.0g of glutamic acid (per liter), adjusted to pH 7.5 (17).

Human and animal sera. Human sera were obtained from patients withwell-documented histoplasmosis (n=18), coccidioidomycosis (n=12), andcandidiasis (n=5). Coccidioidomycosis sera were generously provided byDr. Demothenes Pappagianis, University of California School of Medicine(Davis, Calif.). The histoplasmosis sera were obtained from patientswith acute and chronic disease and from patients with disseminatedinfections (with positive bone marrow and/or blood cultures) associatedwith AIDS. The laboratory diagnosis of histoplasmosis infections wasbased on culture and biopsy results and/or serology tests(immunodiffusion and complement fixation with yeast and mycelialantigens). Blastomycosis sera from humans (n=5) and dogs (n=6) withdocumented clinical infections and sera from rabbits immunized withBlastomyces dermatitidis antigens or whole yeast cells (n=3) were a giftfrom Dr. Gene Scalerone (Idaho State College, Pocatello, Ind.).

Control human sera were obtained from healthy residents of St. Louis,Mo. A histoplasmosis serum pool was prepared with sera (n=12) frompatients with proven histoplasmosis.

Isolation of H. capsulatum DNA. Genomic DNA from yeast cells wasisolated essentially as previously described (17,34). Briefly, yeastcells were pelleted and resuspended in TE buffer (10 mM Tris, pH 8.0; 1mM EDTA). SDS was added to 1% final concentration. DNA was extractedusing phenol-chloroform, ethanol precipitated, and washed with 70%ethanol.

Mouse sera. Antibodies to yeast antigen or to a histidine fusion proteinof the recombinant clone GH17 (GH17-his, see below) were produced in6-week-old female BALB/c mice by foot-pad injection of 10 μg of yeastantigen or purified GH17-his in FCA followed by a second injection ofantigen in IFA 4 weeks later. Sera were collected 1 week after thebooster immunization.

Yeast antigen. Yeast cells were suspended in 0.01M Tris buffer (pH 8.3)containing protease inhibitors (1 mM phenyl methyl sulfonyl fluoride, 1mM EDTA, 25 μg/ml N-tosyl-L-phenylalanine chloromethyl ketone, and 25μg/ml N-a-p-tosyl-L-lysine chloromethyl ketone (all from Sigma ChemicalCompany, St. Louis, Mo.). The yeast homogenate was rocked at 4° C.overnight and centrifuged at 15,000×g for 10 min. The proteinconcentration in the supernatant was measured with a commercial kit(BCA; Pierce Chemical Co., Rockford, Ill.).

Screening of a gene expression library and selection of recombinantclones. A λgt11 cDNA library was custom-synthesized (Clontech Lab. Inc.,Palo Alto, Calif.) using Poly(A)⁺ mRNA derived from the mycelial stageof the G217B strain of H. capsulatum. This library has a recombinantfrequency of over 90% after amplification. The DNA insert size range is0.6-4.5 kb with an average size of 1.6 kb. The library wasimmunoscreened to identify H. capsulatum-specific clones essentially aspreviously described (5,6). Clones that were reactive with antibodies inthe histoplasmosis serum pool but not reactive with a normal human serumpool were selected and purified by repeated cycles of immunoselection.The reactivity of serum pools to fusion proteins expressed by purifiedrecombinant phage was studied by plaque-dot immunoblot analysis aspreviously described (5). PCR was employed to amplify the cDNA insertsof selected recombinant λgt11 clones with the GenAmp DNA amplificationkit (Perkin Elmer-Cetus, Norwalk, Conn.) as previously described (31).DNA dot hybridization was performed using peroxidase-labeled DNAfragments (14) to assess homology between the selected clones.

Southern blot analysis and DNA sequencing. H. capsulatum genomic DNA (5μg) was cut with selected restriction endonuclease enzymes. Digestionproducts were electrophoresed in a 1% agarose gel and transferred toHybond-N+ nylon transfer membrane (Amersham, Arlington Heights, Ill.) bystandard techniques, and blots were probed with labeled cDNA insert ofGH17 (26).

λgt11 DNA purified from GH17 was digested with EcoRI and ligated intopBluescript II SK-(Strategene Cloning Systems, La Jolla, Calif.) bystandard methods (26), and plasmid DNA was prepared for DNA sequencing.The dideoxynucleotide chain termination method (32) was used for doublestranded DNA sequencing using the TaqTrack Sequencing System (PromegaCorporation, Madison, Wis.) with T3 and T7 pBluescript primers and withsynthetic oligonucleotides.

The PC/GENE DNA Sequence Analysis Software (Intelligenetics, MountainView, Calif.) and the BLAST Program (NCBI, NLM, NIH, Bethesda, Md.) wereused to analyze nucleotide and deduced amino acid sequences and todetermine sequence homologies with previously reported sequences in theGenBankm data base.

Expression and purification of GH17 in the pPROEX™−1 Protein Expressionsystem. The cDNA insert of the recombinant clone GH17 was subcloneddirectionally into the plasmid expression vector pPROEX™−1 (Gibco-BRL)to produce a fusion protein containing 6 histidines. GH17 fusion protein(GH17-his) was purified from bacterial lysates by continuous-elutionelectrophoresis using a BioRad Prep Cell (BioRad Laboratories, Hercules,Calif.). Briefly, a 10-ml overnight culture of Escherichia coli (BL21strain) cells containing the recombinant plasmid GH17 was inoculatedinto 700 ml NZCYM medium (Gibco-BRL) containing 50 μg ampicillin per ml(Sigma). Cultures were grown at 37° C. with shaking to OD₆₀₀ of 1.0.IPTG (final concentration, 0.3 mM) was then added, and the culture wasgrown for an additional 5 h, after which the cells were pelleted andresuspended in 1:50 v/v of lysis buffer (10 mM Na₂HPO₄, 30 mM NaCl,0.25% Tween 20, 10 mM EDTA, 10 mM EGTA, pH 7.0). The cells were frozenat −20° C. overnight. Cells were thawed in cold water and lysed by mildsonication. Cellular debris was removed by centrifugation at 10,000×gfor 15 min. A 12% polyacrylamide gel was poured per the manufacturer'sprotocol for the BioRad Model 491 Prep Cell. Ten ml of sample in 1:1loading buffer (0.0625M Tris-HCl, pH 6.8, 10% glycerol, 0.025%bromphenol blue) was loaded and the gel was run for 8 hrs at 12 Wconstant power. Three ml fractions were collected and run on 12%SDS-PAGE minigels (21). Western blots (35) were performed with thehistoplasmosis human serum pool to identify fractions that contained theGH17 fusion protein. Three consecutive fractions containing the band ofinterest were selected, pooled, and dialyzed versus PBS, pH 7.2. Thedialyzed protein was concentrated with a membrane concentrator(Centriplus,™ Amicon, Beverly, Mass.) and the protein concentration wasmeasured with a commercial kit (BCA; Pierce Chemical Co.)

Immunoblot analysis of recombinant fusion proteins. E. coli Y1090 wasinfected at high density with recombinant phage on a thin layer ofagarose over LB-agar to achieve confluent lysis, and synthesis of fusionproteins encoded by cDNA inserts was induced with IPTG-impregnatedfilters. The agarose layer containing bacterial lysate and fusionprotein was then gently scrapped off and dissolved in SDS-PAGE samplebuffer. SDS-PAGE was performed as described by Laemmli (21) at 135 V in8% reducing gels. After SDS-PAGE, proteins were transferredelectrophoretically (35) to nitrocellulose membranes. Membranes werethen incubated in monoclonal antibody to β-galactosidase (PromegaBiotech, Madison, Wis.) or in canine or human sera diluted 1:500 inPBS/T for 3 h at 37° C. Membranes were washed in PBS/T and incubatedwith alkaline phosphatase conjugated goat anti-mouse, anti-dog oranti-human IgG (Promega) for 1 h at 37° C. After washing, membranes weredeveloped with NBT/BCIP.

Immunoblot analysis was carried out with H. capsulatum yeast extract toidentify the native antigen(s) that correspond to the recombinant cloneGH17. Yeast extract was separated by SDS-PAGE on 5-25% gradient slabgels and processed as described above with mouse antibody to GH17-his.

RESULTS

Selection of λgt11 clones that express H. capsulatum-specific antigens.Approximately, 500,000 phage plaques from an H. capsulatum-mycelia phasecDNA expression library were immunoscreened with a histoplasmosis serumpool and a normal control serum pool made from sera obtained fromhealthy residents of St. Louis who had no history of histoplasmosis.Twenty clones selected in the initial screen were rescreened withindividual histoplasmosis sera. Eight highly immunoreactive clones wereidentified. These were again tested by plaque-dot immunoblots with serafrom patients with other non-H. capsulatum fungal infections, includingC. immitis, B. derratitidis, and Candida sp. Four clones that werereactive with antibodies in sera from histoplasmosis patients and notreactive with antibodies from patients with other fungal infections wereselected for further study. DNA dot hybridization studies showed thatall four clones hybridized to each other even under high stringencyconditions (data not shown). The four clones designated as GH2, GH17,GH22 and GH23 produced a similar size β-galactosidase fusion proteinwith an apparent Mr of 140,000 vs. 116 kDa for unfused β-galactosidase(data not shown).

Molecular characterization of recombinant Histoplasma clones. The cDNAinserts of clones GH2, 17 and 22 were sequenced. All three clonescontained identical 5′ ends and an identical 633 bp open reading frame(ORF). That ORF is disclosed herein as SEQ ID NO:2. The three clones hadvariable amounts of untranslated DNA at the 3′ ends (GH2-260 bp;GH17-142 bp; and GH22-166 bp). The complete nucleotide sequence of GH17(GenBank™ Accession number U27588) is disclosed herein as SEQ ID NO:1.The deduced amino acid sequence of the translated protein is disclosedherein as SEQ ID NO:3. The presumed initiation codon 36 bp downstreamfrom the 5′ end is the first ATG in the ORF. The sequence also has apurine (adenine) in the −3 position (Kozak's rule), a prerequisite foran initiation codon (19). The initiation codon is followed by ahydrophobic sequence (predicted by hydropathy analysis, FIG. 2) which isconsistent with a signal peptide sequence. Two potential signalpeptidase cleavage sites were identified by the method of von Heijne(37) which predicts cleavage after residues 20 and 24. The sequence alsocontains a predicted transmembrane helix from amino acid 2 to 28 (28).The 3′ non-coding region has a poly (A) tail of 14 bp. The ORF codes fora protein of 211 amino acids with a predicted size of 23.5 kDa and acalculated pI of 4.15. There are three potential N-glycosylation sitesin the predicted amino acid sequence; these are located in thehydrophilic domains of the protein (boxed areas, FIG. 1). The GH17sequence does not exhibit significant similarity to any proteins presentin GenBank/EMBL sequence databases except for the similarity of thethreonine-rich region to other threonine rich sequences such ascellulase of Caldocellum saccharolyticum (25), Xenopus laevisintegumentary mucin (12), and a Leishmania surface antigen (27) (FIG.1).

Southern blot analysis was performed to identify genomic fragmentscarrying the gene(s) encoding the recombinant clone GH17. When DNA wascut with EcoRI and PstI and probed with labeled cDNA insert of GH17,bands were detected at 4.9 kb and 5.5 kb, respectively (FIG. 3). Theprobe hybridized to two bands (8.5 kb and 5.0 kb) in SacI digested DNA.However, recombinant clone GH17 has an internal SacI site. These resultssuggest a single location in the H. capsulatum genome for GH17.

Sensitivity and specificity of IgG antibodies to recombinant H.capsulatum proteins. Immunoreactivity of recombinant H. capsulatumproteins produced by clones GH2, 17, 22, 23 was assessed by Western blotwith sera from patients with a variety of fungal infections. Most serafrom histoplasmosis patients had easily visible antibody reactivity withall 4 recombinant proteins (Table 1, FIG. 4A). The sensitivity ofWestern blot with these clones for histoplasmosis sera ranged from89-100% (Table 1, FIG. 4A). None of these clones was recognized by serafrom humans and animals infected with other fungi (FIG. 4B).

TABLE 1 Sensitivity^(a) and Specificity of Immunoblot with RecombinantH. capsulatum Clones Clones Serum Source GH2 GH17 GH22 GH23Histoplasmosis Human  18/18^(b) 18/18 16/18 16/18 Blstomycosis Dog 0/60/6 0/6 0/6 Rabbit 0/3 0/3 0/3 0/3 Human 0/5 0/5 0/5 0/5Coccidioidomycosis Human  0/12  0/12  0/12  0/12 Candidiasis Human 0/50/5 0/5 0/5 Uninfected Controls Human  0/12  0/12  0/12  0/12^(a)Immunoreactivity was assessed by immunoblot with β-galactosidasefusion proteins. ^(b)No. of sera reactive/no. of sera tested.

Expression of GH17 in pPROEX™−1 expression vector. The CDNA insert ofGH17 was expressed as histidine fusion in the pPROEX™−1 proteinexpression system. Plasmid pPROEX-1 consists of a Trc promotor for highlevel expression in E. coli, a prokaryotic ribosome binding site, and a6× His affinity tag for ease of purification. A fusion protein with anapparent size of 32 kDa was evident by SDS-PAGE and immunoblot (FIG.5A). GH17-his failed to bind to a metal affinity column. Therefore, thefusion protein was purified from bacterial lysates by continuous-elutionelectrophoresis using a BioRad PrepCell. Western blots were performed toselect fractions of interest by immunoblotting with a humanhistoplasmosis serum pool (FIG. 5B). Three consecutive fractionscontaining the band of interest were selected, pooled and dialyzed. Thisyielded a total of 500 μg of purified protein from approximately 700 mlof bacterial culture.

Pilot studies were carried out to test the purified GH17-his protein inan ELISA format (data not shown). Unfortunately, ELISA based on theGH17-his protein was less sensitive and specific than the recombinantimmunoblot assay with the GH17-β-galactosidase fusion protein. Thislowered specificity and sensitivity is believed to be due tocross-reactivity to the polyhistidine component of the GH17-his protein.

Inmunoblot analysis of mouse antibodies to recombinant antigen. Serafrom mice immunized with GH17-his bound to a 60 kDa native H. capsulatumyeast antigen by Western blot (FIG. 6). This antigen was not recognizedby pre-immune mouse sera.

DISCUSSION

The experiments of this Example demonstrate that H. capsulatum-specificantigens can be identified, cloned, characterized, and produced usingrecombinant DNA methodologies and other methods, as described. Arecombinant H. capsulatum antigen was shown to have highly specific andsensitive immunodiagnostic potential.

Recombinant clones that expressed H. capsulatum-specific antigens wereidentified by several cycles of differential immunoscreening, and themost immunoreactive and specific clone (GH17) was selected for moredetailed studies. GH17 codes for the most promising recombinantdiagnostic antigen for histoplasmosis that has been identified to date.GH17 codes for a protein that corresponds to a 60 kDa native H.capsulatum antigen. There are three potential N-glycosylation sites[Asn-Asn/Lys-Thr] in the predicted amino acid sequence of GH17 (boxedareas, FIG. 1). Glycosylation at these sites could account for thedifference between the predicted polypeptide mass of 23.5 kDa and theobserved size of the native yeast protein recognized by the mouseantibodies to GH17 histidine fusion protein (60 kDa).

The protein encoded by GH17 is highly antigenic in humans withhistoplasmosis. Chou-Fasman predictions based on the deduced amino acidsequence of GH17 (7) indicate that the protein is rich in potential Bcell epitopes. These predictions are based principally on thehydrophilic character and accessibility of highly charged and exposedpolar residues that comprise the turns and alpha helices within thepredicted GH17 protein (13,20). Our results with human sera areconsistent with these predictions. GH17 produced a 140 kDa fusionprotein that was recognized in Western blots by 18 of 18 sera frompatients with histoplasmosis.

In contrast to previous serological work with H. capsulatum (15, 16, 36,40, 41) the GH17 recombinant immunoblot assay appears to have excellentspecificity for histoplasmosis.

As shown in the Example, the present invention provides a method toisolate specific fungal antigens. In particular, the present inventionoffers significant advantages over prior art fungal antigens, antibodiesand diagnostic methods employing the same. The specificity of thepresent target fungus antigens and antibodies make them particularlyadvantageous for reliable determination of the presence of the targetfungus or antibodies which are specific to the target fungus. As appliedto target fungi which are pathogens of vertebrates, such as H.capsulatum, use of antigens and antibodies of the present invention areuseful for providing reliable evidence of present or past infection withH. capsulatum. Other features, objects and advantages of the presentinvention will be apparent to those skilled in the art. The explanationsand illustrations presented herein are intended to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Those skilled in the art may adapt and apply the inventionin its numerous forms, as may be best suited to the requirements of aparticular use. Accordingly, the specific embodiments of the presentinvention as set forth are not intended as being exhaustive or limitingof the invention.

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9 1 810 DNA Histoplasma capsulatum 1 acacagcata acagaagatc tctgaaattgacaaaaatga aaaccatttg cttgcctgct 60 tacttcaagc ttctctcttt tctgtctgctatagctgtga catctggagc agctgttgac 120 tcctgtctct tagaatcaaa ctgcccaccgccaacaacaa caacgacaac aacgacaaca 180 acaccaacac caacaccaac atcaataataccaataacac caatagtacc agcaaataag 240 acaattgtgc ttacaaccac tattgagcctgggccaggcc aggtttgggc gcaaatagag 300 gagattgatc ctgaaccata ttatgttagatgggtccctg atccaacgtt tgccacgcct 360 gttgtactgc acaataacac agatcttgtcttcatggatg gaagcaaatc tttttatctc 420 aacttcgata acagcacctc tgacacgggtatttattttg tgaaccttaa ctccaacgct 480 ggtattagtc aactctataa ggatagtgacaacaagttgc tctggggtgg agctcaacaa 540 gagcgggatg gctggatgtg gtgcttcatggtcgatctac aataccgcat gttctattct 600 gacagtaaat tcgttggttc tccaagggattgtggcctct cctctgtctt tttgacagag 660 cgcccgagtt gaaacagcta ttgtgaggaggggagcagtt ctggaccggc cgtgcgaaat 720 aagtaatgag tatcaaagtg tttctgtgatctatgaaatt tagagggcca ggatacaatt 780 attgatcaac tcccacaaaa aaaaaaaaaa810 2 631 DNA Histoplasma capsulatum 2 atgaaaacca tttgcttgcc tgcttacttcaagcttctct cttttctgtc tgctatagct 60 gtgacatctg gagcagctgt tgactcctgtctcttagaat caaactgccc accgccaaca 120 acaacaacga caacaacgaa caccaacaccaacaccaaca tcaataatac caataacacc 180 aatagtacca gcaaataaga caattgtgcttacaaccact attgagcctg ggccaggcca 240 ggtttgggcg caaatagagg agattgatcctgaaccatat tatgttagat gggtccctga 300 tccaacgttt gccacgcctg ttgtactgcacaataacaca gatcttgtct tcatggatgg 360 aagcaaatct ttttatctca acttcgataacagcacctct gacacgggta tttattttgt 420 gaaccttaac tccaacgctg gtattagtcaactctataag gatagtgaca acaagttgct 480 ctggggtgga gctcaacaag agcgggatggctggatgtgg tgcttcatgg tcgatctaca 540 ataccgcatg ttctattctg acagtaaattcgttggttct ccaagggatt gtggcctctc 600 ctctgtcttt ttgacagagc gcccgagttg a631 3 211 PRT Histoplasma capsulatum 3 Met Lys Thr Ile Cys Leu Pro AlaTyr Phe Lys Leu Leu Ser Phe Leu 1 5 10 15 Ser Ala Ile Ala Val Thr SerGly Ala Ala Val Asp Ser Cys Leu Leu 20 25 30 Glu Ser Asn Cys Pro Pro ProThr Thr Thr Thr Thr Thr Thr Thr Thr 35 40 45 Thr Pro Thr Pro Thr Pro ThrSer Ile Ile Pro Ile Thr Pro Ile Val 50 55 60 Pro Ala Asn Lys Thr Ile ValLeu Thr Thr Thr Ile Glu Pro Gly Pro 65 70 75 80 Gly Gln Val Trp Ala GlnIle Glu Glu Ile Asp Pro Glu Pro Tyr Tyr 85 90 95 Val Arg Trp Val Pro AspPro Thr Phe Ala Thr Pro Val Val Leu His 100 105 110 Asn Asn Thr Asp LeuVal Phe Met Asp Gly Ser Lys Ser Phe Tyr Leu 115 120 125 Asn Phe Asp AsnSer Thr Ser Asp Thr Gly Ile Tyr Phe Val Asn Leu 130 135 140 Asn Ser AsnAla Gly Ile Ser Gln Leu Tyr Lys Asp Ser Asp Asn Lys 145 150 155 160 LeuLeu Trp Gly Gly Ala Gln Gln Glu Arg Asp Gly Trp Met Trp Cys 165 170 175Phe Met Val Asp Leu Gln Tyr Arg Met Phe Tyr Ser Asp Ser Lys Phe 180 185190 Val Gly Ser Pro Arg Asp Cys Gly Leu Ser Ser Val Phe Leu Thr Glu 195200 205 Arg Pro Ser 210 4 25 PRT Caldocellum saccharolyticum 4 Pro ThrSer Thr Val Thr Pro Thr Pro Thr Pro Thr Pro Thr Pro Thr 1 5 10 15 ProThr Val Thr Ala Thr Pro Thr Pro 20 25 5 41 PRT Leishmania 5 Pro Pro ThrThr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr 1 5 10 15 Thr ThrThr Lys Pro Pro Ile Thr Thr Ala Thr Thr Thr Lys Pro Pro 20 25 30 Thr ThrThr Thr Thr Thr Thr Lys Pro 35 40 6 57 PRT Xenopus laevis 6 Thr Thr LysAla Thr Thr Thr Thr Pro Thr Thr Thr Thr Thr Thr Pro 1 5 10 15 Thr ThrThr Thr Thr Thr Thr Thr Thr Thr Lys Ala Thr Thr Thr Thr 20 25 30 Pro ThrThr Thr Thr Pro Thr Thr Thr Thr Thr Lys Ala Thr Thr Thr 35 40 45 Thr ProThr Thr Thr Thr Thr Thr Pro 50 55 7 25 PRT Histoplasma Capsulatum 7 ProPro Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Pro Thr Pro Thr 1 5 10 15Pro Thr Ser Ile Ile Pro Ile Thr Pro 20 25 8 41 PRT HistoplasmaCapsulatum 8 Pro Pro Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Pro Thr ProThr 1 5 10 15 Pro Thr Ser Ile Ile Pro Ile Thr Pro Ile Val Pro Ala AsnLys Thr 20 25 30 Ile Val Leu Thr Thr Thr Ile Glu Pro 35 40 9 57 PRTHistoplasma Capsulatum 9 Thr Ser Gly Ala Ala Val Asp Ser Cys Leu Leu GluSer Asn Cys Pro 1 5 10 15 Pro Pro Thr Thr Thr Thr Thr Thr Thr Thr ThrThr Pro Thr Pro Thr 20 25 30 Pro Thr Ser Ile Ile Pro Ile Thr Pro Ile ValPro Ala Asn Lys Thr 35 40 45 Ile Val Leu Thr Thr Thr Ile Glu Pro 50 55

We claim:
 1. A method for determining the presence or absence of H.capsulatum antibodies in a mammal, the method comprising: obtaining anantibody-containing sample from the mammal, contacting the sample with aprotein antigen of H. capsulatum which has an amino acid sequence thatincludes a portion at least five amino acids in length of the amino acidsequence set forth in SEQ ID NO: 3, and which portion is bound byantibodies to H. capsulatum but which is not substantially bound byantibodies to Coccidioides immitis, Blastomyces dermatitidis and Candidasp., and determining whether an antibody in the sample immunoreacts withthe protein antigen of H. capsulatum.
 2. The method of claim 1 whereinthe mammal is a human.
 3. The method of claim 1 wherein theimmunoreactivity of the antibodies to Coccidioides immitis, Blastomycesdermatitidis and Candida sp. is less than about 10% of theimmunreactivity of the antibodies to H. capsulatum with the proteinantigen of H. capsulatum.
 4. The method of claim 1 wherein theimmunoreactivity of the antibodies to Coccidioides immitis, Blastomycesdermatitidis and Candida sp. with the protein antigen of H. capsulatumis less than about 1% of the immunoreactivity of the antibodies to H.capsulatum with the protein antigen of H. capsulatum.
 5. The method ofclaim 1 wherein the immunoreactivity of the antibodies to Coccidioidesimmitis, Blastomyces dermatitidis and Candida sp. with the proteinantigen of H. capsulatum is less than about 5% of the immunoreactivityof the antibodies to H. capsulatum with the protein antigen of H.capsulatum.
 6. The method of claim 1 wherein immunoreactivity of theantibodies to Coccidioides immitis, Blastomyces dermatitidis and Candidasp. with the protein antigen of H. capsulatum is less than about 2% ofthe immunoreactivity of the antibodies to H. capsulatum with the proteinantigen of H. capsulatum.
 7. The method of claim 1 wherein the proteinantigen of H. capsulatum is substantially free of non-proteindeterminants.
 8. The method of claim 1 wherein the protein antigen of H.capsulatum is substantially free of carbohydrate determinants andphosphorylcholine.
 9. The method of claim 1 wherein the method comprisesan assay selected from the group consisting of a precipitin-basedimmunoassay, an indirect label-based immunoassay, a direct label-basedimmunoassay and an inhibition/competitive-type label-based immunoassay.